Human genome contains ˜600 proteases and homologs, similar to the number of protein kinases (˜500). Proteases are initially described as destructive enzymes that recycle proteins into amino acids. After many years of study, our understanding of protease function has shifted from the nonspecific degradative function to specific proteolytic reactions that regulate protein trafficking, localization, activity and interaction with other proteins, and cleave and activate pro-hormones and peptide neurotransmitter precursors. Proteases therefore regulate many biological processes, including basic molecular processes such as DNA replication and transcription, unfolded protein response and protein degradation; cellular processes such as cell differentiation, proliferation and programmed cell death; tissue morphogenesis and homeostasis such as angiogenesis, neurogenesis, blood coagulation and wound repair. It is thus not surprising that changes to proteolytic systems lead to many diseases including cancer, neurodegenerative and cardiovascular diseases. Many pathogens and viruses also utilize proteases for their invasion and replication in host. In short, proteases play important roles in the development and homeostasis of all living organisms, and are important drug targets.
In view of the important role of proteases, it would be beneficial to monitor, track, and image protease activity. Moreso, it would be ideal to monitor, track, and image protease activity in vivo. One such approach is the design of a protease reporter wherein the protease reporter becomes fluorescent only when activated by a protease. However, there are limitations to the use of known fluoresecent reporters.
For example, although the availability of a wide variety of naturally occurring fluorescent proteins and spectral variants of the proteins has allowed for substantial advances, limitations to the use of fluorescent proteins remain. In particular, the use of fluorescent proteins in intact animals such as mice has been hindered by poor penetration of excitation light. For example, visibly fluorescent proteins have been cloned from jellyfish and corals and have revolutionized many areas of molecular and cell biology through in vivo expression, in vitro expression, protein labeling, and protein engineering; however, the use of such fluorescent proteins for imaging studies in intact animals (e.g., a mouse or human) is limited due to the excitation and emission maxima of the fluorescent proteins.
Specifically, the excitation and emission maxima of these fluorescent proteins generally do not exceeded 598 and 655 nm respectively (D. Shcherbo et al., Nat. Methods 4, 741 (2007); M. A. Shkrob et al., Biochem. J. 392, 649 (2005); L. Wang, W. C. Jackson, P. A. Steinbach, R. Y. Tsien, Proc. Natl. Acad. Sci. U.S.A. 101, 16745 (2004)). One exception are the phytochrome-based fluorescent proteins that have an excitation maximum of 644 nm and an emission maximum of 672 nm (A. J. Fischer, J. C. Lagarias, Proc. Natl. Acad. Sci. U.S.A. 101, 17334 (2004)). However, neither the traditional fluorescent protein cloned from marine animals or the phytochrom-based fluorescent proteins are well equipped for in vivo imaging in whole, living animals.
For example, Green Fluorsecent Proteins are commonly used in fluorescent reporter assays. GFPs are involved in bioluminescence in a variety of marine invertebrates, including jellyfish such as Aequorea Victoria (Morise, H., et al., Biochemistry 13:2656-2662 (1974); Prendergast, F. G., and Mann, K. G., Biochemistry 17:3448-3453 (1978); Ward, W. W., Photochem. Photobiol. Rev. 4:1-57 (1979) and the sea pansy Renilla reniformis (Ward, W. W., and Cormier, M. J., Photochem. Photobiol. 27:389-396 (1978); Ward, W. W., et al., Photochem. Photobiol. 31:611-615 (1980)). The GFP isolated from A. victoria has been cloned and the primary amino acid structure has been deduced. The chromophore of A. victoria GFP is a hexapeptide composed of amino acid residues 64-69 in which the amino acids at positions 65-67 (serine, tyrosine and glycine) form a heterocyclic ring (Prasher, D. C., et al., Gene 111:229-233 (1992); Cody, C. W., et al., Biochemistry 32:1212-1218 (1993)). Resolution of the crystal structure of GFP has shown that the chromophore is contained in a central α-helical region surrounded by an 11-stranded β-barrel (Ormo, M., et al., Science 273:1392-1395 (1996); Yang, F., et al., Nature Biotech. 14:1246-1251 (1996)). Upon purification, native GFP demonstrates an absorption maximum at 395 nm and an emission maximum at 509 nm (Morise, H., et al., Biochemistry 13:2656-2662 (1974); Ward, W. W., et al., Photochem. Photobiol. 31:611-615 (1980)) with exceptionally stable and virtually non-photobleaching fluorescence (Chalfie, M., et al., Science 263:802-805 (1994)).
GFP has been used as a fluorescent label in protein localization and conformation studies and has been used as a reporter gene in transfected prokaryotic and eukaryotic cells (Heim, R., et al., Proc. Natl. Acad. Sci. USA 91:1250-1254 (1994); Yokoe, H., and Meyer, T., Nature Biotech. 14:1252-1256 (1996); Chalfie, M., et al., Science 263:802-805 (1994); Wang, S., and Hazelrigg, T., Nature 369:400-403 (1994)). GFP has also been used in fluorescence resonance energy transfer studies of protein-protein interactions (Heim, R., and Tsien, R. Y., Curr. Biol. 6:178-182 (1996)). Since GFP is naturally fluorescent, exogenous substrates and cofactors are not necessary for induction of fluorescence. Furthermore, the GFP cDNA containing the complete coding region is less than 1 kb and is easily manipulated and inserted into a variety of vectors for use in creating stable transfectants (Chalfie, M., et al., Science 263:802-805 (1994)). However, despite the relative ease at expressing the GFPs, they offer limited use for studying protease activity in vivo.
In vivo optical imaging of deep tissues in animals is most feasible between 650 and 900 nm because such wavelengths minimize the absorbance by hemoglobin, water, and lipids as well as light scattering (F. F. Jobsis, Science 198, 1264 (1977)); R. Weissleder and V. Ntziachristos, Nat. Med. 9, 123 (2003)). Accordingly, the emission maximum of 598 and the absorption maximum of 655 nm of traditional fluorescent proteins (e.g., fluorescent proteins cloned from jellyfish and corals) are ineffective at in vivo optical imaging of deep tissues in animals. Thus, genetically encoded, infrared fluorescent proteins (IFPs) are particularly valuable for whole-body imaging in cancer, stem cell biology, gene therapy, and other areas of biomedical research and treatment.
Furthermore, although many protease-activity based chemical dyes have been developed and applied to the study of disease models, these non-genetically encoded fluorophores are very difficult, if not impossible, to be used in the study of animal development. The fluorescence resonance energy transfer (FRET)-based reporters using GFP and derivatives have been successfully used in detecting protease activity with spatiotemporal resolution in cultured cells. However, the signal of these FRET reporters is weak (several to several tens percent change), which is very challenging in imaging protease activity in animals, due to tissue auto-fluorescence in the visible-wavelength region, cellular heterogeneity and three-dimensional architecture in tissues. It is thus not surprising that FRET sensors are rarely used in whole-animal imaging.
To overcome this problem, other mechanism based reporters have been developed, such as those based on degradation signal or aggregation motif. However, these biological mechanism based reporters are highly dependent on cellular context. For example, the GFP aggregation-based reporter achieves ˜50 fold signal gain in E. coli, but its signal is significantly decreased to only ˜1-3 fold change in mammalian cells [Nicholls, S. B., Chu, J., Abbruzzese, G., Tremblay, K. D., and Hardy, J. A. (2011). Mechanism of a Genetically Encoded Dark-to-Bright Reporter for Caspase Activity. J Biol Chem 286, 24977-24986]. Therefore, a protease reporter based on physical/chemical mechanism would be ideal for its robust use in animals.
In addition the protease reporter being based on a physical/chemical mechanism, an ideal reporter should not be fluorescent until activated by its protease. The novel genetically encoded iProtease described herein is thus an ideal protease reporter, not only because it overcomes the limitations of previously reported chemical dyes and genetically encoded reporters, it is also because its infrared fluorescence is optimized for whole-animal imaging. The rationally designed iProtease provides a general scaffold for further design of many if not all proteases with specific proteolytic activity, and opens opportunity in the study of proteases in animal development and disease. It can also be used in drug development under biological context.